yeast colony PCR - How to improve yeast colony PCR (Dec/26/2007 )

hello,i have promble again.
please give me some suggestion on yeast colony PCR ,this is my protocol
1. Incubate yeast culture with an appropriate medium.at 30°C ,overnight.
2. Centrifuge 0.5ml of cells at 8000rpm for 5mins,discard supernatant.
3. Resuspend the cells with 0.5ml 1xPBS,centrifuge at 8000rpm for 5mins,discard supernatant.
4. Resuspend the cells with 0.1ml TE,then boiled in water for 3 mins
5. cold in the liquid nitrogen for 0.5min
6. boiled in water for 2mins.
7.then take 1ul supernatant as template for PCR

now ,the problem is that i can no get right PCR product ,even get nothing.so i want to know my yest colony protocol is viable or not.

This sounds very complex. If you have a mini-bead beater then breaking the cells with agitation and glass beads would be much easier. A cryotube half filled with small glass beads and some of the culture medium and vortexed vigorously would probably also work. Adding Zymolase to the culture medium for a few minutes, followed by boiling and using 1 ul of the supernatent would be where I would start with chemical techniques. Zymolase works well in lysing the cell wall of yeast. You should also check that your PCR is working with pure DNA.

Is the DNA you are amplifying on a plasmid or chromosomal? If chromosomal, then these chemical and heat/freeze dirty preps will be much less effective.

I don't think the method proposed can sufficiently break down the cell wall. My vote goes to Zymolyase to enzymatically digest the cell wall.

first ,thank you for your suggestions.
i amplied DNA from chromosomal.As you said i need to use Zymolyase to digest the cell wall,right?

If you are amplifying chromosomal DNA, then there are two problems. First, the cells need to be broken open, either mechanically or with Zymolase followed by the normal chemistry (SDS and NaOH, for example). The second problem is that the DNA is mechanically stringy, and needs to be broken up. Mechanical techniques will do this too, but the chemical techniques will leave a stringy mess. You can shear the DNA with a syringe and fine needle, but the whole point here is to do things more easily than a standard DNA prep. I repeat, the easy way is mechanical lysis and DNA shearing with glass beads and vortexing.
http://www.daigger.com/catalog/product/d-B...r%C2%99+Blender
Daigger may not have the best pricing.

phage434,thank you again.i think i konw what you said .but now ,there is a new problem :can i take the supernatant with lysis buffer as template for PCR?

glcui,thanks a lot.i think your suggestion is useful for me though i amplied DNA from chromosomal.